Diverse extracellular cues are transduced through the Ras pathway, and qualitative differences in the duration, amplitude, and subcellular localization of signaling events are critical for achieving the appropriate biological response. These aspects of Ras signaling are influenced by the activities of scaffold proteins that can modulate the activation, function, and/or localization of the core pathway components. In addition, these scaffolds may facilitate cross-talk with other signaling pathways. Over the years, our research on signaling scaffolds has defined a role for the 14-3-3 proteins as key modulators of the active versus inactive conformation of the Raf family kinases and has demonstrated the importance of the KSR family members in the spatio/temporal control of ERK cascade signaling. In addition, our previous work on protein scaffolds identified CNK1 as a positive modulator of Arf GTPase activation and insulin pathway signaling and defined a function for the CNK2 scaffold in the spatial regulation of Rac GDP/GTP cycling during spine morphogenesis in hippocampal neurons. During the reporting period, we have participated in a study identifying KSR1 as a component of perinuclear signaling complexes (PSC) present in tumor cell lines, mouse lung tumors, and mouse embryonic fibroblasts undergoing RAS-induced senescence. I n addition, we initiated a new study in our laboratory to further characterize the function of the Shoc2 scaffold in Ras pathway signaling. Shoc2 was first discovered in genetic screens conducted in Caenorhabditis elegans, where it was identified as a positive modulator of RTK- and Ras-mediated signaling. Subsequently, Shoc2 was reported to function as a regulatory protein for the catalytic subunit of protein phosphatase 1 (PP1) and to play a role in Raf kinase activation. More specifically, binding of the Shoc2/PP1 complex to GTP-bound M-Ras (a relative of the prototypical H-, N- and K-Ras proteins) was found to dephosphorylate a negative regulatory 14-3-3 binding site on the Raf kinases, which promotes Raf binding to the canonical Ras proteins and facilitates ERK cascade activation. Of note, germline mutations in Shoc2 as well as M-Ras, PP1c and the Raf kinases have all been identified as disease drivers in specific subtypes of Noonan syndrome, one of a group of related developmental disorders known as the RASopathies. Our work in this project has been to determine whether the Shoc2 scaffold has additional roles in RTK/Ras signaling that may impact tumor formation and/or contribute to the developmental defects associated with the RASopathies. Towards this end, we have identified a new function for the M-Ras/Shoc2 complex in the dynamic regulation of cell-cell adhesion that is required for collective cell migration. More specifically, we find that M-Ras/Shoc2/ERK cascade signaling plays a role in regulating the p120-catenin/E-cadherin interaction, which in turn controls the cell surface expression and junctional turnover of E-cadherin, a critical component of cell junctions that controls intercellular adhesiveness. Through depletion/reconstitution experiments, these studies also revealed a gain-of-function activity for the RASopathy-associated Myr-Shoc2 mutant. In particular, we found that cells expressing the Myr-Shoc2 mutant or either of two RASopathy-associated C-Raf mutants (S257L or P261S) displayed a less cohesive migratory behavior, which correlated with increased junction turnover and reduced junctional expression of E-cadherin. Expression of Myr-Shoc2 or C-Raf mutants, but not the WT proteins, also induced defects in coordinated convergent/extension cell movements during zebrafish gastrulation, further supporting a regulatory role for the M-Ras/Shoc2/ERK cascade signaling axis in cell migratory events required for normal development. Given the essential role of collective cell migration in human embryogenesis, our elucidation of M-Ras/Shoc2 function in this highly regulated process may provide insight regarding the cellular basis of defects associated with Noonan syndrome and other RASopathies.